Effect of Evodiamine on Cancer Metabolism of Liver Tumor Through Met/EGFR and HIF Pathways

Purpose: To explore whether EVO affects metabolism of liver tumor, and how EVO exert its anti-tumor effect by inhibiting cancer metabolism.Method: CCK8 assay, plate colony forming experiment, transcriptomics and metabolomics assays, flow cytometry, Western Blot analysis, q-PCR technique, lentiviral overexpression gain-of-function experiments were used in vitro experiment, and tumor cells transplantation mice model was used in vivo experiments.Results: EVO markedly inhibited tumor cell proliferation and promoted apoptosis. In the transcriptomic gene chip analysis, EVO suppressed cancer metabolism by down-regulating phosphofructokinase (PFK), PRPP synthase (PRS) and NDP reducase (RR),showed it could restrained glycolysis, PPP and nucleotides synthesis, regulated cell cycle genes transcription and promoted the expression of apoptosis-related genes, and inhibited Met/EGFR, p53-GADD45and HIF pathway, etc. Those results of gene chip were verified by q-PCR and Western Blot. In the result of metabonomics, EVO inhibited pyrimidine nucleotide synthesis, fructose metabolism, active hyaluronic acid, phospholipids, sphingolipid, purine nucleotides synthesis, aspartate, arginine and some other amino acids metabolism. The result of metabonomics assay was consist with Genechip technology. We also used recombinated lentivirus transfection (over-express Met gene) to verify that EVO inhibited the cancer cells growth, RAS-MAPK, PI3K-AKT pathways and cancer metabolism above through inhibiting Met expression, but Met overexpression could not reverse the inhibition of EVO on HIF pathway, indicated that EVO inhibited HIF pathway directly, not through Met. In the experiment of molecular docking, the results showed that EVO had a certain binding energy and a high affinity with Met and EGFR, the intervention of EVO may be through its binding with Met and EGFR. NTP/dNTP could reverse the growth inhibition of EVO, indicated that EVO inhibited the cancer cell growth via inhibiting NTP/dNTP synthesis. But NTP/dNTP could not reverse the growth inhibition of 0.4μM EVO, probably because Evo induced irreversible apoptosis. In vivo experiments, EVO (intragastric administration 50mg/ Kg.d) could significantly reduce the volume and weight of transplanted liver tumor in mice after intragastric administration two weeks, prolonged the survival significantly compared with the control group.Conclusion: EVO may be a specific inhibitor of Met and EGFR. EVO inhibited liver cancer cell proliferation, induced apoptosis and prolonging the survival time of transplanted liver tumor model mice through binding with Met and EGFR so as to inhibit their downstream RAS-MAPK, PI3K-AKT pathways, meanwhile repressing HIF pathways, and then restraining cancer metabolism.Funding Information: This work was financially supported by the National Nature Science Foundation of China (Nos.81774028).Declaration of Interests: The authors declare no conflict of interest.Ethics Approval Statement: Animal experiments were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine.